At the current time, we have a poor understanding of how nontypeable Haemophilus influenzae (NTHi) cause otitis media (OM) in children. We hypothesize: (1) that there are as yet unidentified genes in NTHi that are important in the ability of this organism to cause OM; and (2) that there is a set of genes that are known but whose role in pathogenesis is uncertain or unsuspected. Under current funding, we have begun an in-depth study of NTHi strain 86-028NP pathogenesis in the chinchilla models of OM. Specifically, we have employed differential fluorescence induction using promoter probe constructs; and signature tag mutagenesis to identify strain 86-028NP genes differentially expressed in NTHi and/or genes obligatorily required at one or more stages of the infectious process. We have also sequenced, to 3-fold coverage, the genome of NTHi strain 86-028NP. Some of the genes we identified in NTHi have homology to genes present in the published H. influenzae strain Rd genome. Sequences with no homology to Rd genes but with homology to known genes of other organisms, or unique genes which have no homology to previously identified genes, also have been identified. Although the genome information has been useful and informative, the assembly currently contains 576 contigs, many with regions of low coverage. The current contig data is available on our web site at www.microbial-pathogenesis.org. A global analysis of the current assembly indicates that the gene content and order are similar to that seen in strain Rd. A more detailed analysis reveals that there are a substantial number of genes not previously seen in the Pasteurellaceae and some regions where the gene content and order is different than seen in strain Rd. Thus, the current data suggest that the strain 86-028NP genome will contain a complex mosaic of Rd and non-Rd like features, features that may be important but are not completely discernable from the available data. The tremendous interest in vaccine development for NTHi disease, the increase in our understanding of the pathogenesis of NTHi disease and the knowledge that will be gained from the comparative genomics of different members of the Pasteurellaceae family makes it imperative that we have the complete genome sequence of at least one pathogenic NTHi strain. The National Institute on Deafness and Other Communication Disorders (NIDCD) has been extremely supportive of the functional studies of strain 86-028NP (R01DC03915 from NIDCD/NIH, Lauren Bakaletz, PI) as well as the funding to obtain the 3-fold genome sequence coverage of this economically important pathogen (supplement to R0I DC03915 from NIDCD/NIH). We propose to close and annotate the genome of nontypeable H. influenzae strain 86-028.